Detection of specific nitrated markers

ABSTRACT

Methods are described for improving the diagnostic possibilities of diseases where oxidative NO-modifications occur, for example inflammatory conditions, cancer, Parkinson&#39;s or Alzheimer&#39;s disease, and to provide means of monitoring the effects of therapeutical measures taken towards such diseases. The invention enables the detection of disease specific catabolic markers related to oxidative NO-modifications, utilizing an immunoassay comprising antibodies directed against nitrated and non-nitrated epitopes characteristic of a specific protein.

This application is a divisional application of U.S. patent applicationSer. No. 10/507,479, filed Sep. 13, 2004, now U.S. Pat. No. 7,393,649,which is a nationalization of PCT/EP03/02559 filed Mar. 12, 2003,published in English, which claims benefit of U.S. ProvisionalApplication 60/363,925 filed Mar. 13, 2003.

The present invention relates to a method for performing a qualitativeor quantitative assay which may be used for detecting quantifying ormonitoring oxidative damage especially in relation to an inflammatorycondition. Detection of nitrated specific proteins or fragments thereofversus equivalent non-nitrated proteins or fragments thereof, may serveas an index for oxidative damage in for example inflammatory boweldisease, systemic lupus erythematosus, arthritis, cancer, Parkinson's orAlzheimer's disease.

Throughout a lifetime organisms are challenged with numerous events andconditions that generate reactive oxygen species (ROS). An increase inan organism's rate of ROS production or a decrease in their rate ofscavenging will increase the oxidative modification of cellularmolecules, including DNA and proteins. Oxidation may have deleteriouseffects on protein function and stability. Many enzymes have been shownto lose their biological activity as a consequence of oxidation. Othereffects of oxidation are lowered temperature-stability and changes inprotein susceptibility towards proteolysis; the latter might lead to anaccumulation of oxidized proteins unable to undergo degradation. Proteinoxidation may be implicated in the pathogenesis of several diseases suchas neurodegenerative diseases, cancers, arteriosclerosis,cataractogenisis, dysplasia, dystrophia and inflammatory diseases aswell as in normal ageing.

Some of the common ROS generating processes and systems known to modifyproteins are irradiation, inflammation, metal catalysed reactions suchas Fe(II) or Cu(I) reduction and various other oxidizing compounds orfree radicals, including nitric oxide (NO), peroxy-itrite, H₂O₂ orhydroxyl, hydroperoxyl superoxide and lipid peroxyl radicals. A numberof ROS are formed by specific enzyme systems such as nitric oxidesynthetase (NOS), cyclo-oxygenase and mono-amine oxidase B, whereof someare induced under inflammatory conditions.

Under normal conditions the oxidative potential in the micro-environmentof an organism is under tight control by a number of balancing systemsincluding antioxidants, free radical scavengers, reductases,peroxidases, catalase, glutathione-S-transferase, super-oxide dismutaseand various metal-binding proteins. These systems can be viewed asprotection mechanisms, more than repair systems. Actual repairmechanisms specific for oxidative damaged proteins are rare, whereas theoxidative damage to nucleic acids is subject to highly efficient repairsystems (Stadtman and Levine 2000).

Nitric oxide (NO) is produced in many tissues and regulates diversefunctions, such as smooth muscle relaxation, non-specific defenseagainst microorganisms, neurotransmitter and a possible modulator of thecartilage matrix. Nitric oxide synthetase (NOS) is responsible for theproduction of NO. There are two classes of NOS, a constitutive (cNOS)and an inducible (iNOS) form. iNOS activity appears in response tovarious cytokines, and produces a much larger amount of NO than cNOS.iNOS activity is thought to account for the proinflammatory effects ofNO, as seen in conditions such as inflammatory bowel diseases,spontaneous gut inflammation, cardiovascular inflammation and arthriticdiseases (osteoarthritis (OA) or rheumatoid arthritis (RA)).

The large cytotoxicity of NO is partly due to its ability to react withsuperoxide anion (O₂ ⁻) to generate peroxynitrite anion (ONOO⁻) and itsconjugate acid, peroxynitrous acid (ONOOH). At neutral pH ONOO⁻ ispartly protonated, generating ONOOH, which rapidly decomposes tonitrate. These strong oxidants might seriously compromise cellularregulation, as it is capable of nitrating aromatic compounds like freephenylalanine, tyrosine and tryptophan as well as peptide chainscontaining these amino acids. This result in nitrophenylalanine,nitro-tryptophan and nitrotyrosine, the later can also be generatedthrough the combined hydroxylation and nitration of a phenylalanineresidue (Lin et al 2000). The nitration is irreversible and inhibits thephosphorylation of tyrosine and tryptophan residues, thus interferingwith signal transduction pathways.

In OA and RA, NO is produced in large amounts by chondrocytes,macrophages and inflamed synovium. A high level of nitrite/nitrate hasbeen found in the synovial fluid, serum and urine of patients with OAand RA (Lotz 1999). However, elevated NO levels cannot be considered aspecific marker for any given disease or condition, as several differentprocesses and tissues can give rise to systemic elevated NO levels.

The major clinical manifestation of RA as well as OA is an abnormal anddegraded cartilage. However, until now it has been difficult to directlyassess the ongoing cartilage destruction in arthritis patients, becausespecific markers for this process have not been available in theclinical practice. At clinical diagnosis of OA and RA, damage tocartilage in joints is recorded by X-ray, which reveals a loss of jointspace as cartilage is destroyed and lost. Furthermore the patients arescored according to the pain and mobility problems caused by the jointdestruction, but even though a number of standardised rating systemshave been introduced, it is difficult to quantify these parameters.Other markers used for assessment of RA patients, such as C-reactiveprotein and Rheumatoid factors are associated with the inflammatoryprocess involved in the disease, but are probably not directly relatedto the level of cartilage destruction and they are not specific for RA.

Detection of metabolites, such as cartilage oligomeric matrix protein(COMP), hyaluronates, aggrecan and collagen type II or III fragmentsarising from destruction of joints affected by inflammatory disease havebeen reported (Moller 1998, Wollheim 1996, U.S. Pat. No. 5,919,634, U.S.Pat. No. 6,132,976 and PCT application WO 01/38872). The clinicalusefulness of these markers, however, remains to be proven.

The detection of NO₂-modified amino acids is known from the PCT patentapplications WO 96/04311 and WO 98/29452. These patent applicationsdisclose the sequence independent detection of a nitrotyrosine or anitro-tryptophan residue in a protein or in its free form using anantibody, which specifically recognizes the nitro-group. Such anantibody might be used to assess a pathological condition relating to anabnormal level of nitrotyrosine. However the antibody will not be ableto assess the problem in relation to a specific tissue or protein as itrecognizes nitrotyrosine independent of the surrounding amino acidsequence.

The present invention relates to methods for quantifying NO₂-modifiedamino acids within the specific context of a protein or fragmentsthereof. Determination of such nitrated specific proteins or fragmentsthereof enables an assessment of oxidative damage and metabolic state ofthe given protein. This provides diagnostic assays, which associatemetabolic changes and oxidative damage in specific diseases.

According to the present invention there is provided a method forperforming a qualitative or quantitative assay for protein oxidation,comprising detecting in a sample an amino acid sequence which ischaracteristic of a specific protein and which contains one or morearomatic amino acid residues in nitrated form. This includes a methodfor detecting oxidative damage in a mammal. The method may comprisemonitoring oxidative damage by detecting one or more nitrated aromaticresidues in combination with an amino acid sequence specific to aprotein or peptide, preferably specific to a certain tissue, in abiological sample. Preferred aromatic amino acid residues arenitrotyrosine and/or nitrotryptophan.

The method of the present invention can be applied for monitoring apathological process involving an oxidative damage correlating withnon-inflammatory diseases like arteriosclerosis, cancer, Alzheimer'sdisease, Parkinson's disease or inflammatory diseases like asthma,cardiovascular inflammation, diabetes, inflammatory bowel disease,psoriasis, systemic lupus erythematosus, arthritis.

The method of the present invention will enable the monitoring of acatabolic process of a joint tissue by measuring a protein or peptidederived from the extra cellular matrix of cartilage, joint synovium orsubchondral bone. Such a protein might be collagen types I, II, III, VI,IX or XI, aggrecan, biglycan, chondromodulin, cartilage link protein,cartilage oligomeric matrix protein, cartilage intermediate layerprotein or a fragment thereof, wherein a nitrotyrosine and/ornitrotryptophan residue is located.

The detection performed in the method of the present invention may becarried out using an antibody, which specifically binds a nitratedepitope comprising at least one nitrated aromatic amino acid residue inconjunction with a specific amino acid sequence.

The detection performed in the method of the present invention can alsobe carried out utilizing two antibodies, one which is specific for thenitrated aromatic amino acid residue in a context independent manner andone which recognizes the specific amino acid sequence.

The detection methods of the present invention can be performed in a waythat generate an index of oxidative damage/inflammation, by using asecond antibody, which specifically binds a non-nitrated amino acidsequence equivalent to the nitrated sequence bound by the antibody whichrecognizes the nitrated epitope described above, thereby generating aratio between a specific nitrated and non-nitrated protein or peptide.

Alternatively, the relative amounts of nitrated and non-nitrated proteinor peptide may be differently expressed, for instance as a difference oras the ratio of the nitrated content to the total of nitrated andnon-nitrated content.

The index of oxidative damage/inflammation generated by application ofthe present invention, to a sample from a mammal, can be used to providemeans for diagnosis or assessment of the severity of a disease involvingoxidative damage, especially joint tissue diseases. For these purposes akit utilizing an antibody, which recognizes a nitrated epitope and asecond antibody recognizing the similar non-nitrated epitope, togetherwith suitable labels, is provided. A supplement to such a kit is apeptide, which contains a succession of amino acids equivalent to thebinding epitope for one of the mentioned antibodies, for competitionassays. The kits of the present invention can be applied to samples likemammalian body fluids, extracts from cells or tissues or supernatantsfrom cells or tissues cultured in vitro.

The present invention especially relates to detection of nitratedcollagen type II protein, wherein the nitrated amino acid is thetyrosine of one of the sequences His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly SEQID NO: 1 or Leu-Gln-Tyr-Met-Arg-Ala SEQ ID NO: 2. Synthetic nitratedpeptides including these sequences may be used to raise polyclonaland/or monoclonal antibodies, as well as cell lines producing suchmonoclonal antibodies.

The present invention is based upon a new approach for development ofdiagnostic and prognostic assays for monitoring pathological conditionsin of mammalian tissues, combining metabolic tissue specific markerswith the state of an oxidative/inflammatory condition. One of the keyeffects of oxidative damage is nitration of aromatic amino acid residuessuch as phenylalanine, tyrosine or tryptophan. The presence of one ormore nitrated aromatic amino acid residues in or near a marker specificto a certain tissue or disease, provides information about the metabolicstate of a tissue from which a marker originates as well as theoxidative condition of the same tissue.

As used herein, “immunological binding partner” includes polyclonal,monoclonal or humanized antibodies, including Fc fragments, Fabfragments, chimeric antibodies or other antigen-specific antibodyfragments.

As used herein “biochemical marker” or just “marker”, includes aprotein, protein fragment, polypeptide, domain structure, peptide orotherwise proteolytically processed protein, representing changes withina specific tissue, which becomes detectable in relation to such changes.

As used herein, “nitrated epitope”, includes a site within an antigencontaining a nitrophenylalanine, nitrotyrosine or nitrotryptophanresidue, where the epitope recognized by an antibody constitutes thenitrated residue and enough adjacent amino acid residues to gain proteinspecificity for the antibody.

As used herein “two independent epitopes”, means two sites within thesame protein, polypeptide, or peptide recognized by two differentantibodies which preferably can bind their respective epitopessimultaneously. Preferably one of the sites is or contains a singlenitrated aromatic amino acid residue.

As used herein, “nitrated aromatic amino acid residue”, means aphenylalanine, tyrosine or tryptophan residue situated in a protein orpeptide, where the aromatic ring of the amino acid residue has beenmodified by covalent attachment (substitution) of a NO₂-group.

As used herein, “nitrophenylalanine” means a phenylalanine residuesituated in a protein or peptide, where the aromatic ring of thephenylalanine residue has been modified by covalent attachment of aNO₂-group.

As used herein, “nitrotryptophan”, means a tryptophan residue situatedin a protein or peptide, where the aromatic ring of the tryptophanresidue has been modified by covalent attachment of a NO₂-group.

As used herein, “nitrotyrosine”, means a tyrosine or phenylanalineresidue situated in a protein or peptide, where the aromatic ring of thetyrosine has been modified by covalent attachment of a NO₂-group or thearomatic ring of the phenylalanine residue has been modified by combinedcovalent attachment of an OH-group at position 4 and a NO₂-group in oneof the remaining positions.

As used herein, “Tyr:NO₂”, means nitrotyrosine. The preferred positionof the nitro-group is adjacent to the hydroxyl-group, for example asshown in Formula I.

In one embodiment of the present invention a protein, peptide orfragment thereof containing a nitrated aromatic amino acid residue isdetected by means of identification, in which the identification relieson detection of the nitrated aromatic residue, in combination with aspecific amino acid sequence. The nitration of the aromatic amino acidhas arisen as a result of oxidative damage, preferably connected to aninflammatory condition.

In one preferred embodiment of the present invention the nitratedaromatic amino acids to be measured are nitrotyrosine and/ornitrotryptophan residues. Most preferred are nitrotyrosine residues,which can either be generated through the nitration of a tyrosineresidue, or through the combined hydroxylation and nitration of aphenylalanine residue.

In pathological processes involving oxidative damage, the degree ofnitrated proteins can be used to assess the severity of this damage.

In one embodiment of the present invention this is assessed bygenerating an index of oxidative damage by measuring a nitrated residuein conjunction with its surrounding sequence of a protein, peptide orfragment thereof containing a nitrated aromatic amino acid residueversus the equivalent non-nitrated protein, peptide or fragment thereof,with the exception that a nitrotyrosine may be a phenylalanine in thenon-nitrated peptide.

Another embodiment of the present invention includes its application forin-vitro diagnosis or assessment of severity of a disease connected toan oxidative pathology and/or inflammatory condition. Such diseasescould be, but is not limited to, arteriosclerosis, Alzheimer's, asthma,Crons disease, Parkinson's disease, cancer, cataractogenisis, diabetes,bronchopulmonary dysplasia, multiple sclerosis, muscular dystrophy,inflammatory bowel diseases, psoriasis, systemic lupus erythematosus,osteoarthritis or rheumatoid arthritis

Inflammation of joint tissues is seen in conjunction with arthriticdiseases such as RA and OA or as a result of an acute joint injury. Thisinflammatory condition is very often connected to a catabolic processwithin the tissue, leading to its gradual degradation. Such catabolicprocesses within a tissue, releases proteins, peptides or fragmentsthereof to numerous body fluids, like the synovial fluid, blood, serumor urine. Such molecules can be utilized as biochemical marker(s) forjoint tissue degradation/catabolism. Protein or protein fragmentsderived from the cartilage matrix or subchondral bone, containing atleast one tyrosine, phenylalanine or tryptophan residue are more likelyto become nitrated upon the inflammatory increase in NO production asseen in OA and RA. Measuring nitrated versus non-nitrated catabolicmarkers enables the correlation between degradation and inflammation.This principle can also be applied to markers for gut inflammatorydiseases or other diseases where oxidative NO₂-modifications occur, suchas in arteriosclerosis, cancer, Alzheimer's or Parkinson's disease.

In one embodiment of the present invention the nitrated aromaticresidues have arisen as a result of an inflammatory condition in a jointtissue is situated in a protein, peptide or fragment thereof derivedfrom the extra cellular matrix or cartilage, joint synovium orsubchondral bone, which can be measured in order to monitor thecatabolic process in the tissue.

In one preferred embodiment, the protein or fragment thereof, which ismonitored, constitutes a marker of cartilage degradation associated withan inflammatory joint disease. The nitrated form versus the non-nitratedform of such a cartilage matrix derived marker is measured generating anindex of inflammation.

Proteins or fragments thereof, which can be nitrated and act as markersof interest include, but are not limited to, collagen types I, II, III,VI, IX or XI, aggrecan, cartilage link protein, cartilage oligomericmatrix protein, cartilage intermediate layer protein.

A preferred marker protein is collagen type II. Collagen type IIcontains two tyrosine's, which can be nitrated upon oxidative damage.The first sequence HRGYPGLDG (His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly) SEQID NO: 1 is localised in the triple helical region while the secondsequence LQYMRA (Leu-Gln-Tyr-Met-Arg-Ala) SEQ ID NO: 2 is located in thenon-helical domain at the C-telopeptide. The amino acids sequencesincluding the tyrosine residues are specific for type II collagen andcan be employed as specific biochemical markers of catabolic processesin the cartilage tissue.

A preferred embodiment is the detection of the nitrated type II collagenmarker versus the equivalent non-nitrated type II collagen markerproviding an index of the oxidative damage and/or the inflammatorycondition of the cartilage tissue in associated with the metaboliccondition of the same tissue. The principle of this method also appliesto other tissue specific markers originating from oxidative damagedtissues undergoing metabolic changes at the same time.

Especially for monitoring the results of a treatment this is ofimportance, as some forms of treatment might influence the catabolicprocess whereas others might influence the inflammatory state. This moredifferentiated assessment of the disease obtained by application of thepresent invention will enable therapeutic interventions to be targetedto individual patients.

The detection of a nitrated aromatic amino acid residue in combinationwith a sequence located within a specific protein, peptide or fragmentthereof can be performed in numerous ways, such as, but not limited to,HPLC, mass spectroscopy, iso-electric focusing, sequencing orimmunoassays.

One preferred method of detection is the use of an immunoassay,utilizing an antibody, which specifically binds at least one nitratedaromatic amino acid residue in conjunction with the surrounding aminoacid sequence of a specific protein (nitrated epitope). Assay forms inwhich such an antibody can be applied include, but not limited to,ELISA, microarray, RIA, FACS, Western blotting, immunoaffinitychromatography, and immuno-histochemistry.

Another method of detection is a sandwich immunoassay, utilizing anantibody, which specifically recognizes a nitrated aromatic amino acidresidue independent of the surrounding sequence and a second antibody,which recognizes a specific amino acid sequence located within the sameprotein, peptide or a fragment thereof as the nitrated amino acidresidue. The second antibody can very well be a polyclonal antibody withspecificity towards a specific protein e.g. collagen type II, where theactual epitope has not been identified.

The most preferred method for monitoring a pathological processinvolving oxidative damage and/or an inflammatory condition inassociation with metabolic changes utilizes the generation of an indexas described above. More specifically such an index is generated bycontacting an antibody, which specifically binds a nitrated epitope,with a biological sample. Upon reaction with the first antibody, theantibody-peptide conjugates can be detected by differentaffinity/visualization or isolation methods. If appropriate, a secondantibody, which specifically binds an equivalent non-nitosylated aminoacid sequence as the first antibody, is contacted with an aliquot of thesame biological sample as the first antibody, or the remnants(supernatant) resulting from an isolation of the first antibody-peptideconjugate. The ratio between nitrated and non-nitrated peptide and/orprotein in the biological sample is determined. Methods of determinationare well known in the art, for example ELISA, microarray, RIA, FACS,immunoaffinity chromatography, and immunohistochemistry. If applyingother methods than chromatography, it is important that the twoantibodies are labelled in a manner that enables differentiation betweenthem. This could be different fluoresce (e.g. red, green, yellow),enzymatic label vs. radioactive label and so forth. An index ofoxidative damage and/or inflammation in association with the metaboliccondition of the tissue from where the specific protein originates canbe provided by evaluating ratios from a patient in relation to ratiosfrom healthy individuals.

In situations where a tissue sample is used for monitoring ofpathological processes in joint tissue, there is a strong likelihoodthat denatured helical collagen domains, resulting from catabolicprocesses within the tissue, might be retained in the tissue bycross-linking and fibrillar packaging. To address this problem, thebiological sample is first contacted with an enzyme having the abilityto selectively cleave unwound (non-helical) collagens without cleavingthe His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 1 and/or theLeu-Gln-Tyr-Met-Arg-Ala SEQ ID NO: 2 epitope. Such enzymes could be, butis not limited to, trypsin or chymotrypsin, which are unable to cleavewound (native) collagen within the α-helix. The fragments of unwoundcollagen are then extracted from the biological sample to produce anextract of unwound collagen fragments. This extract can then be assayedas mentioned in the above.

Antibodies with the properties described above are raised against apeptide constituting a nitrated epitope. The peptide is used as anantigen for immunisation. The peptide is emulsified in an adjuvantmedium, preferably incomplete Freund's adjuvant and injectedsubcutaneously or into the peritoneal cavity of a mammalian host,preferably a rodent most preferred rabbits, even more preferred BalbCmice. To enhance immunogenic properties of the antigenic peptide it canbe coupled to a carrier protein before emulsified in an adjuvant medium.Useful carriers are proteins such as keyhole limpet hemocyanin (KLH),edestin, thyroglobulin, albumins, such as bovine or human serum albumin(BSA or HSA), tetanus toxoid, and cholera toxoid, polyaminoacids, suchas poly-(D-lysine-D-glutamic acid). Booster injections may be given atregular intervals until an immune response is obtained, the lastinjection may be given intravenously to ensure maximal B-cellstimulation.

Antisera will be screened for their ability to bind the desired epitopeand their amount of cross reactivity to the non-nitrated epitope.Antisera from the most promising hosts may be used in their crude formor purified.

Monoclonal antibodies may be prepared from the immunised mice with thehighest antibody titre, by fusing lymphocytes isolated from the spleenof these mice with a myeloma cell line. The generated hybridoma clonesare screened for their ability to produce antibodies, which recognizethe desired epitope. Cell lines can be established for production andpurification of monoclonal antibodies.

Methods for polyclonal and monoclonal antibody production are well knownin the art and other methods than the described can also be utilized.

In one aspect of the present invention the synthetic peptide forantibody and cell line generation as described above is(X_(aa))_(m)-His-Arg-Gly-Tyr:NO₂-Pro-Gly-(X_(aa))_(n) SEQ ID NO: 3wherein X_(aa) denote any amino acid or derivatives thereof and m and nare independent integers e.g. from 0 to 10.

In one preferred embodiment of the present invention the syntheticpeptide for antibody and cell line generation as described above is(X_(aa))_(m)-His-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly-(X_(aa))_(n) SEQ IDNO: 4 wherein X_(aa) denote any amino acid or derivatives thereof and mand n are independent integers e.g. from 0 to 10.

In another preferred embodiment the synthetic peptides for antibody andcell line generation as described above has the form(X_(aa))_(m)-Leu-Gln-Tyr:NO₂-Met-Arg-Ala-(X_(aa))_(n) SEQ ID NO: 5wherein X_(aa) denote any amino acid or derivatives thereof and m and nare independent integers e.g. from 0 to 10.

With the exception that a nitrotyrosine may be derived from aphenylalanine in the non-nitrated peptide, the second antibodiesutilized in the present invention are generated using the same orsimilar techniques as for the preparation of the nitrosyl bindingantibodies.

One embodiment of the present invention constitutes the development of adiagnostic kit for use in detection and monitoring of oxidative damageand/or an inflammatory condition. This includes an antibody recognizinga nitrated epitope, preferably utilizing an antibody of the presentinvention, either alone or in combination with a second antibody withspecificity towards the equivalent non-nitrated sequence, enabling asimultaneous assessment of tissue metabolism and oxidative damage. Thekit can be applied on mammalian body fluids or extracts of cells ortissues, preferably derived from humans. For competition detections apeptide of 6 to 20 amino acids, in which a succession of amino acids isequivalent to the binding epitope for one of said antibodies, might besupplied either in a labelled or non labelled form. The antibodies maybe labelled by joining them, either covalently or non-covalently, with areporter molecule. Suitable reporter molecules or labels, which may beused for ease of detection, include radioisotopes, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like. One of thenon-labelled antibodies or a peptide of the kit might be immobilised,preferably on a solid surface like a micro-titter plate, possibly byconjugation to a suitable protein carrier like BSA.

In a preferred embodiment the first antibody in the kit described aboverecognize the nitrosylated collagen type II sequences previouslydescribed, and the second antibody recognizes the equivalentnon-nitrated sequence.

The invention will be further described and illustrated with referenceto the accompanying drawings, in which:—

FIG. 1 shows a standard curve for nitrated collagen type II immunoassayin a semi-logarithmic plot. The concentration of free antigen is in nM.B/Bo represents the ratio between antibody bound to coated antigen inthe presence of free antigen (B) or in the absence of free antigen (Bo)and is given in percentage;

FIG. 2 shows competitive inhibition of antiserum D37 binding toHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 6 coated plates usingHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly(◯), SEQ ID NO: 6His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly(), native type II collagen (♦),nitrated type II collagen (⋄), type I collagen ( ), BSA ( ) and nitratedBSA (∇) as competitors. B/Bo represents the ratio between antibody boundto coated antigen in the presence of competitor antigen (B) or in theabsence of competitor antigen (Bo) and is given in percentage;

FIG. 3 shows results from Example 3 and gives the level of Coll2-1(NO2)fragments in serum from Lewis rats immunized with bovine collagen typeII. The graphs depict the levels of the markers in animals which did notdevelop disease (CIA healthy) and the animals that developedinflammatory arthritis (CIA sick);

FIG. 4 shows results from Example 4 demonstrating that human articularcartilage explants produce Coll2-1(NO2) in vitro. From left to right thebars show results from a control, upon stimulation with IL1, oncostatinand plasminogen, and upon stimulation with IL1, oncostatin, plasminogenand the plasminogen activator aminophenyl mercuric acetate (APMA);

FIG. 5 shows results obtained in Example 5 giving the physiologicalvariation of Coll2-1 (NO2) fragment levels in serum according to agerange and sex (men plain black diamonds, and women white diamonds);

FIG. 6 shows results obtained in Example 5 giving the physiologicalvariation of Coll2-1(NO2) fragment levels in serum in men and womensubdivided in two large age groups;

FIG. 7 shows results obtained in Example 6 demonstrating that RApatients have more elevated Coll2-1 (NO2) levels than OA patients; and

FIG. 8 provides results obtained in Example 7 showing levels of crossreactivity of antibody anti-Coll2-2 (NO2) (antiserum D33).

EXAMPLES Example 1 Collagen Type II Immunoassay Antisera:

A sequence of nine amino acids (His-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly)SEQ ID NO: 6 derived from the triple helical region of type II collagen[(α1) II] and a second sequence of six amino acidsLeu-Gln-Tyr:NO₂-Met-Arg-Ala SEQ ID NO: 7 derived from the C-telopeptideof type II collagen were synthesized using standard Fmoc solid-phasepeptide synthesis (HBTU/HOBt protocol) (Chan, W. C. and White, P. D.,2000).

The amino acids sequence was conjugated to thyroglobulin by acarbodiimide procedure (Soinila et al 1992).

Rabbits were injected intraperitoneally with 1 ml of the conjugateemulsified in complete Freund's adjuvant. The conjugate and the adjuvantwere mixed in equal volumes. Injections were repeated four times everymonth with a similar amount of conjugate in incomplete Freund'sadjuvant. Ten days after the last injection, the rabbits were sacrificedfor the final bleeding. Blood were collected and centrifuged for 10minutes at 1500×g at 4° C. The supernatants were stored at −20° C.

The following examples will concentrate on antisera achieved fromimmunisation with the His-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO:6 peptide. All examples can be performed in similar ways for theLeu-Gln-Tyr:NO₂-Met-Arg-Ala SEQ ID NO: 7 peptide.

Six antisera, identified as Coll2-1:NO2 D35, D36, D37, D38 D39 and D40,were obtained and their specificity were tested with the competitiveinhibitions His-Arg-Gly-Tyr(NO₂)-Pro-Gly-Leu-Asp-Gly, SEQ ID NO: 6His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 1, type II nitratedcollagen, native type II collagen, type I nitrated collagen I, type Icollagen, nitrated BSA and BSA.

Competitive ELISA:

A competitive immunoassay was developed to quantify breakdown productsof nitrated type II collagen containing following sequenceHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 6. SyntheticHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 6 peptides werebiotinylated and incubated at 1.25 ng/ml on streptavidie coated plates(Nunc, Denmark) for 1 hour at room temperature. Fifty μl of calibrators(to generate a standard curve) or unknown samples, diluted in Ultroser G(Gibco) were added to separate wells. Hundred μl antiserum (see above)diluted 1/125000 was added to each well. Samples were mixed by rotatingthe plate and incubated 1 hour at room temperature. After threesuccessive washings with washing buffer (Tris 25 mM, NaCl 50 mM pH 7.3),100 μl of horseradish peroxidase-conjugated goat antibodies to rabbitIgG (Biosource, Belgium) were added to each well and incubated 1 hour atroom temperature. After washing, 100 μl of freshly prepared enzymesubstrate (TMB, Biosource, Belgium) were added to each well. After 15minutes incubation, the reaction was stopped with 100 μl 4M H₃PO₄. Thecoloration was read with a microplate reader (Labsystem iEMS Reader MF,Finland) at 450 nm and corrected for absorbance at 620 nm. A standardcurve was constructed on a log-linear graph by plotting the B/Bo of 6calibrators (10 to 0.01 nM) (FIG. 1). The concentration ofHIS-ARG-GLY-TYR:NO₂-PRO-GLY-LEU-ASP-GLY SEQ ID NO: 6 containing peptidesin the samples, were determined by interpolation on the calibrationcurve.

Detection Limit

The detection limit of the assay described in example 1, is calculatedas the mean (M) Bo value of 21 determinations of standard A minus 3times the standard derivation (SD) of Bo (M_(A)−3*SD_(A)). ForColl2-1:NO2 D37 the detection limit was 25 pM.

Example 2 Characterisation of Antisera Coll2-1:NO2 D37-40 Specificity

The antisera produced, were tested for their specificity forHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 6, by use of theimmunoassay described in example 1. To test for specificityHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 6,His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 1 peptide, type IInitrated collagen, native type II collagen, type I nitrated collagen,type I collagen, nitrated BSA and BSA.

Native type II collagen, type I collagen, nitrated collagen type I,nitrated BSA and BSA, were not able to compete with the coatedHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO:6 peptide in theapplied concentrations, whereas the antiserum showed weak affinity tothe non-Nitrated His-Arg-Gly-Tyr-Pro-Gly-Leu-Asp-Gly sequence SEQ ID NO:1 and nitrated collagen type II and strong affinity to theHis-Arg-Gly-Tyr:NO₂-Pro-Gly-Leu-Asp-Gly SEQ ID NO: 6 sequence. A lack ofbinding affinity has also been demonstrated with L-nitrotyrosine.

Example 3 Coll2-1(NO2) Levels are Elevated in Animal Models ofInflammatory Arthritis

A number of animal models have been established to study inflammatoryarthritis. These models generally reflect the human disease, by havingjoint inflammation and tissue destruction. Significant up-regulation ofinflammatory cytokines and NO levels have been detected. Thus,nitrosylation of tyrosine residues is likely to occur in thesesituations. By this example, it was demonstrated that sera samples ofnormal rats and mice could be measured in the Col2-1 (NO2) assaydescribed in example 1.

One of the most commonly employed animal models of RA is the Lewis-ratCollagen Induced Arthritis (CIA) animal model. In this model, jointinflammation is induced by immunization with collagen type II whichprovoke a severe inflammatory response to joint cartilage apparent aspaw joint swelling and subsequent destruction of joints whenhistological analysis is performed. Arthritis was monitored bymacroscopic scoring of swelling and redness of the paws. Cartilage andbone erosion was monitored by quantifying urinary levels of CartiLapsand serum levels of RatLaps, two biochemical markers of cartilage andbone resorption. These assays were performed as specified by themanufacturer (Nordic Bioscience Diagnostics, Herlev, Denmark).

The study cohort comprised 21 female Lewis rats which are ovariectomized(OVX). At baseline, the weight was determined and the animals wereanesthetized for the OVX procedure. After induction of generalanesthesia with Hypnorm-Dormicum (1 part Hypnorm®+1 part Dormicum®+2part sterile de-ionised water, dose 0.15-0.2 ml/100 g body weight), astandard OVX was performed. The weight of the animals was determined ona weekly basis throughout the study period. For measurements of bone andcartilage markers urine samples were obtained by either placing theanimals in a metabolic cage for 30-60 min and awaiting spontaneousurination or by gently rubbing the belly of the animal, i.e. forcedurination and by obtaining blood samples as eye-blood. At studytermination, the knees were isolated and the articular cartilage wasanalyzed for erosion histologically. The rats were immunized withcollagen type II 1 week after OVX to induce inflammatory arthritis. Eachrat was immunized with 150 μg bovine collagen type II emulsified inIncomplete Freunds Adjuvant. The paws of the rats were scored daily fromday 11 by visual observation of the paws. Each paw is scored on a scale0-4 and the score for the four paws are added. If the combined arthritisscore reached a score of 10 for a given animal or if the animaldeveloped very severe arthritis in one paw or had CIA for more that 16days, the animal was sacrificed for ethical considerations, as moresevere arthritis is associated with significant pain and discomfort. Theremainder of the animals were maintained for 27 days after OVX beforebeing terminated.

The Coll2-1 (NO2) level in the serum and the presence of external signsof arthritis had a very good correlation (FIG. 3). The levels ofColl2-1(NO2) were significantly elevated in the rats which developedinflammatory arthritis (sick) whereas animals which did not developdisease showed significantly lower levels of Col2-1(NO2). Moreover, thelevel of collagen fragments increased before the appearance of severearthritis symptoms implying that Coll2-1(NO2) is a predictive biomarkerfor inflammation induced cartilage erosion.

Example 4 Human Articular Cartilage Explants Produce Coll2-1 (NO2) inVitro

Articular cartilage explants are commonly used as an in vitro system toassess cartilage metabolism. Articular cartilage was obtained from adulthuman patients undergoing joint replacement surgery and the cartilagewas excised either as cylindrical plugs (5-30 mg) or as slices (20-30mg). The explants were cultured in 96-well plates in 200 mL serum freeDMEM medium, in the presence of recombinant human IL-1α5 ng/mL (Sigma,St. Louis, USA), Oncostatin M 50 ng/mL (Sigma, St. Louis, USA) and humanplasminogen 10 μg/mL (Sigma, St. Louis, USA). Plasminogen is aphysiological MMP activator that induces collagen type II degradation.Furthermore the MMP activator APMA (aminophenyl mercuric acetate, SIGMA,St Louis, USA) was added as indicated in FIG. 4. The conditioned mediumwas harvested at various time points for measurement of Coll2-1(NO2)This example shows how the cytokines IL1 and oncostatin (OSM) influencethe release of Coll2-1(NO2) in the conditioned medium from cartilageexplants.

It was demonstrated that addition of IL1, oncostatin and plasminogen hadno influence on cartilage degradation. However a significant level ofColl2-1(NO2) could be detected in conditioned medium of cartilageexplants when the plasminogen activator APMA was also added to themedium (FIG. 4). This observation links the release of the Col2-1(NO2)marker from articular cartilage to collagenolytic activity in thematrix, and demonstrates that the marker reflects catabolic processes inthe tissue.

Example 5 Physiological Levels of Coll 2-1(NO2) in Healthy Men and Women

To establish reference values for Coll 2-1 (NO2), sera were collectedfrom 242 healthy ambulatory subjects attending a blood donor centre inLiege, Belgium. None of the study subjects had any evidence of arthritisor other inflammatory disease. None was currently taking any medicationknown to modify arthritic disease or influence joint metabolism. Thisgroup was composed by 170 men and 72 women, aged from 20 to 65 years(mean: 42.8±1.4 years). The mean age of women was 42.7±1.0 years old andthe mean age of men was 42.8±1.4 years old.

When the population was stratified by age in 5 years brackets, Coll 2-1(NO2) serum levels did not vary significantly in the investigated ageinterval (20-65 years) (FIG. 5). The comparison of peptide levels by sexshowed that up to 45 years of age, Coll 2-1 (NO2) concentration washigher in women than in men but the difference was only significant forthe subclasses 20-25 and 26-31 years old (figure X+3). However, whensubjects aged from 46 to 55 years corresponding to the earlypostmenopausal women were removed, Coll 2-1 (NO2) level was higher inpre-menopausal women than in post-menopausal women.

Coll2-1(NO2) level is higher in young individuals (pre-menopausal) thanin older individuals (FIG. 6). The level of these fragments is identicalfor both men and women in the range of 56-65 years old whereaspre-menopausal women showed significantly higher levels of the markercompared to men and postmenopausal women. This observation correspondswell with the established link between estrogen levels and nitric oxide.Estrogen up-regulates production of inducible nitric oxide synthetase(iNOS) and thus levels of nitric oxide, which in turn is responsible forgeneration of nitro tyrosine modified proteins as quantified in theCol2-1(NO2) assay. Hence the elevated levels of this marker measured inthe premenopausal women establishes a link between estrogen levels andnitrosylation.

Example 6 RA Patients have More Elevated Coll2-1(NO2) Levels than OAPatients

An important clinical issue is whether levels of the Col2-1(NO2) markerare elevated in arthritis and are associated with the inflammatoryprocess seen in RA. To study this, serum samples were obtained from across-sectional panel of arthritis patients comprising 10 OA patients (4women and 6 men aged over 45 years) who were candidates for arthroscopy.Arthroscopy was performed for diagnosis and/or shaving of the meniscusand cartilage lesions. Sera were collected 24 hours prior surgery. Thesesubjects had no radiological signs of OA but all had cartilage lesionsidentified by arthroscopy. All subjects had a normal leukocytosis and aC-reactive protein (CRP) level inferior to 5 mg/L. Furthermore, thesepatients did not take any nonsteroidal anti-inflammatory drugs duringthe year before the intervention.

Coll 2-1 (NO2) concentration were also measured in serum samples of 14patients with early RA. At the sampling time, these patients had notreceived any medication, and all had a C-reactive protein level above 5mg/L.

When comparing the levels of Coll2-1(NO2) in these cohorts it wasapparent that the RA patients where inflammation of the joint tissue isa significant element of the disease had significantly higher levels ofthe marker than OA patients or controls (FIG. 7). This observationassociated the Coll2-1(NO2) production directly to inflammationassociated cartilage degradation in inflammatory arthritis.

Example 7 Development of an Assay Specific for a Nitrosylated CollagenType II Epitope Derived from the C-Telopeptide Region (Coll2-2(NO2)ELISA) Reagents and Buffers for Immunoassays

The coating buffer was 0.08 M NaHCO₃ pH 9.6. The saturation buffer wascomposed of 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, 2 mM KCl, 138 mM NaCl, 5 g/Lbovine serum albumin (BSA), 53 g/L lactose monohydrate pH 7.2. Thewashing buffer was a solution of 25 mM Tris, 50 mM NaCl pH 7.3. Thestandard curve and the dilution of samples, when it was necessary, wererealized in 50 mM Tris, 138 mM NaCl, 7 g/L BSA, 1 ml/L Tween 20 pH 8.0.

The dilutions of the antisera ( 1/80 000) and of the second antibody1/5000) were done in Na₂HPO₄ 10 mM, KH₂PO₄ 1.5 mM, KCl 2 mM, NaCl 150mM, EDTA 25 mM, BSA 1% Tween 0.1% pH 7.4.

Immunization

Rabbits were injected intra-peritoneally with 1 ml of the conjugatedpeptides (0.5 mg/ml) emulsified in complete Freund's adjuvant. Theconjugate and the adjuvant were mixed in equal volumes. Injections wererepeated four times every month using the same peptide concentrationthat those of the first injection in incomplete Freund's adjuvant. Tendays after the last injection, the rabbits were sacrificed. Blood wascollected and centrifuged for 10 min at 2500 rpm at 4° C. Thesupernatant was kept and stored at −20° C. At each bleeding, antiserawere screened by titration experiment for the presence ofanti-GGGLQY(NO₂)MRA SEQ ID NO:8 antibody. The antisera with the highesttiters were selected for the following experiments.

Specificity

D33 antibody is highly specific for nitrosylated Coll2-2 fragment anddoes not react with the same fragment non-nitrosylated Coll2-2 or withany other fragment slightly similar in the sequence (Coll2-1) orcompletely different sequences (BSA NO2). The figured showed also thatthe chosen sequence of Coll2-2 against which D33 was raised cannot berecognized when it is still “included” in the full length protein (CollII (NO2)). The nitrosylated sequence is recognized only in the form offree fragments (FIG. 8).

Assay Technical Performance:

CV intra-assay: In order to address the question of correct andreproducible results measured in this assay intra-assay CV was evaluatedby measurement of the same urine samples in different emplacement in thesame plate.

TABLE 1 Evaluation of intra-assay CV (%) of Coll2-2 NO2) assay. Coll 2-2(NO2) concentration CV intra-assay (Mean ± SD, nM) (%) Urine 1 1.56 ±0.15 9.1 Urine 2 2.86 ± 0.26 9.1

The intra-assay showed that the samples could be measured in a goodreproducible manner (Good standard variation of samples meanconcentration of Coll2-2(NO2) fragments). The CV on samples measurementfor both samples used here were below 10% witnessing also of the assayprecision.

Recovery: The certainty of Coll2-2 measurement is assessed by successivedilution of the samples. The dilution of the samples allows us toevaluate the recovery (expressed in %). The table below summarize theevaluated recoveries for two urine samples. The assay performedcorrectly (see table below).

TABLE 2 Recovery of Coll2-2 (NO2) fragments in two urine samples afterdilution of respectively ½; ¼ and ⅛. Coll 2-2 (NO2) concentration, nMRecovery (%) Urine samples 1 Undiluted 9.26 — Dil. ½ 5.57 120.1 Dil. ¼2.26 106.0 Dil. ⅛ 1.42 122.3 Urine samples 2 Undiluted 2.74 — Dil. ½1.55 113.1 Dil. ¼ 0.85 124.1 Dil. ⅛ 0.33  95.6

REFERENCES

-   Chan, W. C., White, P. D., 2000 Fmoc solid-phase peptide synthesis:    A practical approach, Oxford University Press, Oxford 2000.-   Lin, J. K., Chen, K. J., Liu, G. Y., Chu, Y. R., Lin-Shiau, S.    Y., 2000. Nitration and hydroxylation of aromatic amino acid and    guanine by the air pollutant peroxyacetyl nitrate. Chem Biol    Interact. 127, 219-236.-   Lotz, M., 1999. The role of nitric oxide in articular cartilage    damage. Rheum Dis Clin North Am 25, 269-282.-   Moller, H. J., 1998. Connective tissue markers of rheumatoid    arthritis. Scand J Clin Lab Invest 58, 269-278.-   PCT patent application WO 96/04311, Ye Y. Z, Beckman J. S.,    Monoclonal Antibody to Nitrotyrosine, Methods for Diagnosis and    Methods for Treatment of Disease, University Alabama Res Found (Us),    1996.-   PCT patent application WO 98/29452, Chagnaud J. L., Vincendeau P.,    Geffard M., Veyret B., Antibodies Specifically Recognizing a    Nitrated Protein, Method of Preparation, Therapeutic and Diagnostic    use, Centre Nat Rech Scient (Fr), 1998-   PCT patent application WO 01/38872, Christgau S., Henriksen D. B.,    Cloos P., Assay of Isomerised and/or Optically Inverted Proteins and    Protein Fragments, Osteometer Biotech A/S (DK), 2001.-   Soinila, S., Mpitsos, G. J., Soinila, J., 1992. Immunohistochemistry    of enkephalins: model studies on hapten-carrier conjugates and    fixation methods. J Histochem. Cytochem. 40, 231-239.-   Stadtman, E. R., Levine, R. L., 2000. Protein oxidation. Ann. N. Y.    Acad. Sci. 899, 191-208.-   U.S. Pat. No. 5,919,634, Eyre D. R., Methods of detecting collagen    type II degradation in vivo, Washington Research Foundation    (Seattle, Wash.), 1999.-   U.S. Pat. No. 6,132,976 Poole A. R., Hollander A. P.,    Billinghurst R. C., Immunoassay For the Measurement of Collagen    Denaturation and Cleavage in Cartilage, Shriners Hospital for    Children (Tampa, Fla.), 2000.-   Wollheim, F. A., 1996. Predictors of joint damage in rheumatoid    arthritis. APMIS 104, 81-93.

1-26. (canceled)
 27. A method for performing an immunoassay comprisingthe steps of: detecting binding of an immunological binding partnerwhich is immunoreactive with one or more aromatic amino acid residues innitrated form of an amino acid sequence which is characteristic of aspecific protein.
 28. The method as claimed in claim 27, wherein saidimmunological binding partner is specifically reactive with saidnitrated form of said aromatic amino acid residue in the context of saidamino acid sequence which is characteristic of a specific protein. 29.The method as claimed in claim 28, wherein said immunological bindingpartner is reactive with said nitrated form of said aromatic amino acidresidue in a context independent manner and said assay comprisesdetecting binding of both said immunological binding partner and asecond immunological binding partner to nitrated amino acid sequences ina sandwich format, wherein said second immunological binding partner hasbinding specificity for an amino acid sequence which is characteristicof said specific protein.
 30. The method as claimed in claim 27, whereinsaid specific protein of which the detected nitrated amino acid sequenceis detected is a mammalian protein.
 31. The method as claimed in claim30, wherein said protein is present in joint tissue.
 32. The method asclaimed in claim 31, wherein said protein is collagen of type I, II,III, VI, IX or XI, aggrecan, cartilage link protein, cartilageoligomeric protein, or cartilage intermediate layer protein.
 33. Themethod as claimed in claim 27, wherein said nitrated aromatic amino acidresidue or residues is/are nitrotyrosine or nitrotryptophan.
 34. Themethod as claimed in claim 33, wherein the amino acid sequence which isdetected comprises the sequence HRGYPGLDG in which the amino acidresidue Y is nitrated tyrosine or is comprised within said sequence andincludes said nitrated tyrosine.
 35. The method as claimed in claim 33,wherein the amino acid sequence which is detected comprises the sequenceLQYMRA in which the amino acid residue Y is nitrated tyrosine or iscomprised within said sequence and includes said nitrated tyrosine. 36.An immunological binding partner specifically reactive with the nitratedform of an aromatic amino acid residue in the context of an amino acidsequence which is characteristic of a specific protein.
 37. Theimmunological binding partner as claimed in claim 36, having bindingspecificity for an epitope contained in the amino acid sequenceHRGY:NO₂PGLDG which epitope contains the amino acid residue Y:NO₂ and ischaracteristic of collagen type II.
 38. The immunological bindingpartner as claimed in claim 37, which is an antibody raised against apeptide having the sequence (X_(aa))_(m)HRGY:NO₂PGLDG(X_(aa))_(n),where-in X denotes any amino acid or derivative thereof and m and n areindependent integers of from 1 to 10, or is a fragment of such anantibody.
 39. The immunological binding partner as claimed in claim 36,having binding specificity for an epitope contained in the amino acidsequence LQY:NO₂MRA which epitope contains the amino acid residue Y:NO₂and is characteristic of collagen type II.
 40. The immunological bindingpartner as claimed in claim 39, which is an antibody raised against apeptide having the sequence (X_(aa))_(m)LQY:NO2MRA(X_(aa))_(n), whereinX denotes any amino acid or derivative thereof and m and n areindependent integers of from 1 to 10, or is a fragment of such anantibody.
 41. The immunological binding partner as claimed in claim 36,which is a monoclonal antibody or fragment thereof.
 42. A cell lineproducing a monoclonal antibody or fragment thereof as claimed in claim41.
 43. A method for the investigation of the existence or extent of apathological state comprising measuring in a biological sample therelative amounts of nitrated an non-nitrated forms of an amino acidsequence which is characteristic of a specific protein and whichcontains one or more nitratable aromatic amino acid residues.
 44. Themethod as claimed in claim 43, wherein the pathological state isoxidative damage associated with an inflammatory joint disease and saidspecific protein is derived from cartilage matrix.
 45. The method asclaimed in claim 43, wherein said pathological state is a cancer,Alzheimer's disease, Parkinson's disease, an inflammatory bowel disease,systemic lupus erythematosus, osteoarthritis or rheumatoid arthritis.46. The method as claimed in claim 45, comprising: contacting abiological sample from an individual or a portion of such a sample witha first immunological binding partner which binds said nitrated form ofsaid amino acid sequence and quantitatively determining said binding;contacting the biological sample or a portion thereof with a secondimmunological binding partner which binds said non-nitrated form of saidamino acid sequence and quantitatively determining said binding;determining a ratio between said determinations to provide a ratio ofthe relative amounts of said nitrated and non-nitrated forms of saidsequence present in the sample; an comparing the measured value withvalues characteristic of healthy individuals or individuals of knownpathology.
 47. The method as claimed in claim 46, wherein said firstimmunological binding partner is an immunological binding partnerspecifically reactive with the nitrated form of an aromatic amino acidresidue in the context of an amino acid sequence which is characteristicof a specific protein.
 48. A kit for use in performing a method asclaimed in claim 27, and comprising: an immunological binding partnerwhich is specifically reactive with the nitrated form of an aromaticamino acid residue in the context of an amino acid sequence which ischaracteristic of a specific protein; and means for detecting binding ofsaid binding partner and said protein or a fragment thereof.
 49. The kitas claimed in claim 48, wherein the immunological binding partner is animmunological binding partner specifically reactive with the nitratedform of an aromatic amino acid residue in the context of an amino acidsequence which is characteristic of a specific protein.
 50. The kit asclaimed in claim 48, including an immunological binding partner which isreactive with the said amino acid sequence in non-nitrated form.
 51. Thekit as claimed in claim 48, comprising a peptide reactive with a saidimmuno-logical binding partner.